The islets of Langerhans, endocrine tissue containing insulin producing beta cells, represent about one to two percent of the total mass of the human pancreas. Separation or isolation of the islets from the connective matrix and remaining exocrine tissue is advantageous and beneficial for laboratory experimentation and transplantation purposes. Islet transplantation is a most promising and minimally physiologically invasive procedure for treatment of type I diabetes mellitus. Transplanting islets rather than complete pancreatic tissue has the distinct advantages of ease of transplantation, and the elimination of the pancreatic exocrine function of the donor tissue involving secretion of digestive enzymes. Liberating islets from pancreatic exocrine tissue is the initial and crucial step that influences islet transplantations. The important objective in islet isolations is to provide sufficient numbers of viable functional and potent islets for transplantation.
Early methods of islet separation involved chopped pancreatic fragments mixed with collagenase and incubated around 37 degrees C, D. Scharp, World Journal of Surgery 8:143–151, 1984, incorporated herein by reference. Collagenase breaks down and digests pancreatic tissue, freeing islets, however, prolonged exposure to proteolytic enzymatic digestion destroys the initially separated islets. If the separation is stopped to protect the early released islets, too few islets are freed from the surrounding exocrine tissue. Islet isolation in small mammals was improved utilizing mechanical distension of the pancreas, increasing islet yield through mechanical separation of pancreatic tissue. Yet, with larger mammals this technique did not allow sufficient numbers of islets to be separated from a single donor for transplantation. Further improvements in islet separation included perfusion of the pancreas with collegenase via ductal distention causing mechanical disruption of exocrine tissue, U.S. Pat. No. 5,322,790, D. Scharp, Jun. 21, 1994, incorporated herein by reference. In addition, tissue disintegration resulting in islet separation by mechanical disruption of the pancreas has been effected by perfusion of the pancreas with an enzyme containing solution while in a chamber with solid spheres and contacting (beating) the pancreas with the spheres, either by hand or by motorized repetitive motion. This is mentioned in U.S. Pat. No. 5,853,976, Hesse, et al., Dec. 29, 1998, incorporated herein by reference, and is a component of the standard isolation technique currently practiced. Recently, islets have been isolated using enzymatic digestion and sound waves to rupture pancreatic tissue, U.S. Pat. No. 5,879,939, Gray et al., Mar. 9, 1999, incorporated herein by reference. Islets have also been separated using both a warm and cold digestion stage, in which physiologic solutions other than Hanks Balanced Salt Solution (HBSS) have been utilized for the second cold digestion stage, with increased islet yield and functionality when compared to the islets separated utilizing HBSS for the second cold separation, U.S. Pat. No. 5,919,703, Mullen, et al., Jul. 6, 1999, incorporated herein by reference.
While larger scale islet separation from human pancreases has become possible with advances in technology, the previously cited techniques fall short in terms of efficiency, and are inadequate for scale up or mass production in which many donor pancreases are processed at different research and transplantation centers, medical facilities, or commercial locations. In consideration of the lack of donor pancreases, current islet isolation techniques are also inadequate to continuously and repetitively batch process porcine pancreases, or pancreases from animals or mammals, transgenic or non-transgenic, to produce islets for xenotransplantation. In the previously referenced techniques and patents there exist limitations in the methodologies that may significantly affect the outcome of the islet separation process.
Collegenases, metalloendoproteinases that cleave collagen into smaller peptide fragments, are zinc-containing enzymes that require divalent calcium as a cofactor for stabilization and optimal activity. Using traditional collegenases detrimentally affects pancreatic digestion due to the impurities present in the collegenase solutions. Traditional collagenase preparations are concentrated from bacterial (Clostridium histolyticum) culture supernatants. Roche, a manufacturer of molecular biochemicals, states that such collegenase preparations are heterogeneous, containing as many as 30 different enzymes, pigments, cellular debris, and endotoxins. The most significant liabilities of traditional collegenase are variability and endotoxin levels. In traditional collagenase, the primary enzymatic constituent is collegenase, classes I and II, described by Bond and Van Wart, Biochemistry, 23:3077, 1984, and Biochemistry, 23:3085–3091, 1984, incorporated herein by reference. Other proteases found include neutral protease, clostripain, elastase, trypsin, and aminopeptidase. Non-proteolytic enzymes isolated from collagenase preparations include hyaluronidase, galactosidase, acetyl glucosaminidase, phospholipase, fucosidase, and neuraminidase.
Endotoxin is associated with a number of cellular events including cell activation with subsequent cytokine secretion, and programmed cell death (apoptosis). Endotoxin exposure is postulated to cause a loss of transplanted pancreatic islets, Vargas et al., Transplantation, 65(5): 722–727, Mar. 15, 1998, incorporated herein by reference. They have demonstrated that supernatants generated during islet separation were able to induce certain inflammatory cytokines in the islets during the separation process. They postulate that endotoxins and locally induced cytokines accompanying the transplanted islets activate the endothelium and promote lymphomonocytic infiltration of the transplanted islets and surrounding liver tissue. Jahr et al., J. Mol. Medicine (Berlin), 77(1):118–120, January 1999, incorporated herein by reference, suggest that endotoxin-induced early inflammatory reactions may inhibit the function and survival of isolated cells or cell aggregates after transplantation. Eckhardt et al., J. Mol. Medicine (Berlin), 77(1):123–125, January 1999, incorporated herein by reference, have determined that islet xenograft survival increased in endotoxin free conditions. Clearly, endotoxin and accompanying cellular reactions may cause non-function and rejection of transplanted islets.
Alternatively, Roche manufactures non-traditional purified collegenase blends of collegenase I and II (including either neutral protease dispase or neutral protease thermolysin) for pancreatic tissue dissociation, under the product name of Liberase®. Liberase prepared for human islet separation incorporates thermolysin. Neutral protease acts synergistically with collegenase during intercellular matrix digestion. During Liberase enzyme production, collagenase isoenzymes are purified from raw collagenase by a process that removes greater than 99% of the endotoxin present in the collagenase raw material. Enzyme purification also removes non-enzymatic components and bacterial cellular debris that may be toxic to islets. Collagenases I and II are purified to greater than 95% homogeneity. Roche proof data shows that Liberase contains on average less than 10 Endotoxin Units (EU) per milligram (mg), while traditional collegenase preparations contain far greater than 1000 EU/mg and may have as much as 13000 EU/mg. Liberase represents a defined product for tissue dissociation, U.S. Pat. No. 5,952,215, Dwulet, et al., Sep. 14, 1999, incorporated herein by reference. Linetsky et al., Transplant Proc., 30(2):345–346, March 1998, incorporated herein by reference, demonstrated that the use of Liberase enzyme improved human islet yield, compared with traditional collagenase. Examination of absolute islet number, islet number per gram of pancreas, islet equivalent number, and islet equivalent number per gram of pancreas indicated that Liberase enzyme improved islet yield. Liberase gives maximal tissue dissociation performance with minimal endotoxin complicity when compared with traditional collegenases.
Apoptosis can also be initiated by biochemical factors other than endotoxin. Nitric oxide and its metabolites are known to cause cellular death from nuclear damage (apoptosis), U.S. Pat. No. 5,834,005, A. Usula, Nov. 10, 1998, incorporated herein by reference. Nitric oxide is a recognized multifunctional mediator that is produced by and acts on various cells, and participates in inflammatory and autoimmune-mediated tissue destruction, U.S. Pat. No. 5,919,775, Amin et al., Jul. 6, 1999, incorporated herein by reference. The group of enzymes known as nitric oxide synthases catalyzes nitric oxide production. Nitric oxide synthase (NOS) is expressed in mammalian cells. Utilizing cofactors in the presence of oxygen, it catalyzes the mixed functional oxidation of L-arginine to L-citrulline and nitric oxide, by removing a guanidino nitrogen from L-arginine to form nitric oxide. Interleukin-1 (IL-1) has been shown to induce the expression of the cytokine inducible isoform of nitric oxide synthase in pancreatic islets. The production of nitric oxide has been proposed to be the effector molecule that mediates IL-1's inhibitory effects on islet function, U.S. Pat. No. 5,837,738, Williamson et al, Nov. 17, 1998, herein incorporated by reference.
Yet, the deleterious effects of nitric oxide on islet cells can be alleviated by a variety of means. Inhibitors of nitric oxide synthase have been identified. Nitric oxide synthase (NOS), and subsequently nitric oxide, can be inhibited by derivatives of L-arginine, the natural substrate of nitric oxide synthase. These include methyl-, dimethyl-, or amino-substituted guanidines. These inhibitory compounds are also chemically known as aminoguanidinie, N,N′-diaminoguanidine, methylguanidine and 1,1-dimethylguanidine (U.S. Pat. No. 5,837,738 and U.S. Pat. No. 5,919,775, both previously incorporated herein by reference). Nitric oxide production can also be inhibited by 2,4-diamino-6-hydroxy-pyrimidine, a compound that interferes with the activity of a cofactor of inducible nitric oxide synthase. Antibiotic tetracycline also inhibits nitric oxide synthase, thus preventing the formation of nitric oxide, as do doxycycline, and minocycline, a semisynthetic tetracycline (U.S. Pat. No. 5,919,775, previously incorporated herein by reference). Nitric oxide can also be inhibited by nitric oxide scavengers such as cysteine, and other sulfated compounds such as dextran, heparin, and cystine, U.S. Pat. No. 5,834,005 (previously incorporated herein by reference). Alternatively, sparging with an inert gas such as helium can effectively control and eliminate the dissolved oxygen concentration in the islet containing physiologic process solution, thereby hindering the production of nitric oxide via reduction and catalytic oxidation of L-arginine by NOS and cofactors. In combination with oxygen removal from the process solution, cysteine, dextran, heparin, and cystine also inhibit nitric oxide formation that results from relative states of islet hypoxia. Nitric oxide inhibition and scavenging improves islet survival and secretory function. It is certainly beneficial to control or inhibit the formation of nitric oxide in islets and the islet containing physiologic process solution.
The method of mechanical tissue dissociation with glass marbles, steel balls or other sufficiently dense and solid objects, either by hand or with mechanical shaking (Ricordi shaker), may cause tissue damage and trauma to islets resulting from excessive shear stress during the separation process. While repetitive mechanical agitation and contacting the pancreas with solid objects effects tissue disruption aiding enzymatic digestion, such current practices in standard isolation techniques are subjective, and vary between research facilities and transplantation centers.
Although sonication has been employed to aid pancreatic tissue digestion, one certain limitation in this technique is the ‘static’ water bath that the ‘bagged’ pancreas is placed in. Interestingly, this technique is continued until the pancreas appears ‘cracked’, yet, no mention of the internal temperature of the pancreas is noted. Static digestion by any method offers no means of forced-convective heat transfer to maintain a constant processing temperature (cooling during sonication) of the digesting pancreas or the resulting tissue suspension, by the process solution. It is possible that the internal temperature of a bagged pancreas in such a static system exceeds 37 to 40 degrees C., a temperature considered optimal for functional enzymatic digestion, yet, minimal in thermal shock and deactivation of islets due to elevated temperature. A statically digested pancreas in a bag offers no opportunity to maintain a controlled internal pancreatic temperature. This method presents no opportunity to dilute the tissue suspension, which precludes a real-time method to control, deactivate, or inhibit the digestive enzymes in the processing solution, during islet separation and processing, in the dilution and collection phase.
At the XVIII International Congress of the Transplantation Society, Aug. 27–Sep. 1, 2000 in Rome, Italy, advances in pancreatic islet cell transplantation procedures were reviewed and discussed. Existing limits of transplantation and novel approaches to achieving tolerance were evaluated. It was noted that success of recent transplantations (Edmonton Protocol) might certainly be due to the use of immunosuppression that was not toxic to beta cells. Avoiding the use of corticosteroids, induction therapy with anti-IL-2 antibody, and low-dose tacrolimus and sirolimus maintenance were undoubtedly key factors in non-rejection and continued islet tolerance. The quality of the purified islets also contributed to the success of the transplantations, yet, acquired by tedious and laborious manual methods lacking in process control methodology and neglecting important process variables. Current challenges were also assessed, specifically, the standardization of islet separation technology, and the need to development a standardized, reproducible, and automated method to separate and produce high-quality islet cells.
Presently there exists no process control methodology of the islet separation process that takes into account crucial process variables that may be controlled to optimize islet isolation while standardizing and automating the islet separation process. Importantly, there are separation and processing variables that have been neglected and omitted which compromise the reproducibility and repeatability of the islet separation process from location to location. Objectively applying advanced process control methodology and automating the islet isolation process with process control technology can optimize the islet separation process.